Hyper-sensitivity related gene

ABSTRACT

The hsr203J gene of SEQ ID No. 1 and individual components thereof including its promoter and regulatory regions thereof, its coding region, its gene product; modifications thereto; applications of said gene, promoter region, regulatory region and coding region and modifications thereto; DNA constructs, vectors and transformed plants each comprising the gene or part thereof.

This invention relates to an hsr (hypersensitivity-related) gene familyand individual components thereof including its promoter and regulatoryregions thereof, its coding region, its gene product; modificationsthereto; applications of said gene, promoter region, regulatory regionand coding region and modifications thereto; DNA constructs, vectors andtransformed plants each comprising the gene or part thereof.

The hypersensitive reaction (HR) of higher plants is a local inducibleresponse associated with disease resistance to a pathogen. This responseis characterized by a rapid and localized necrosis of tissues invaded byan incompatible (avirulent or non-host) pathogen, which prevents furtherspread of the invading microorganism. Several defense genes whoseproducts may intervene in this plant response have been extensivelystudied: they include enzymes of the phenylpropanoid pathway involved inthe synthesis of antimicrobial phytoalexins, enzymes with hydrolyticactivities, toxic compounds and cell wall proteins. In infected plants,these genes are induced around the necrosis, once it has developed, i.e.late during the HR. Moreover, most of them are also strongly expressedduring compatible interactions leading to the disease of the plant, andfor some of them, during the normal development of the plant. The lackof specificity of these defense genes as well as their activation in thelate steps of the HR suggest that they may not account by itself forestablishment of the complex inducible response that is the HR, butrather may accompany this reaction. To date, the molecular mechanismsleading from plant-pathogen recognition to development of the HR are notknown. In the "gene for gene" hypothesis, the initial step ofplant-pathogen recognition leading to resistance involves the putativeinteraction between the products of a plant resistance gene and of thecorresponding pathogen avirulent gene. Genetic studies indeed revealedthat the outcome of many plant-pathogen interactions are determinedthrough single dominant genes in both partners. Several rapidphysiological changes have been also associated with the HR, such aselectrolyte leakage, changes in respiration rates and more recentlyoxidative cross-linking of cell-wall proteins. However, in no case has aplant gene been described whose activation is specific or at leastpreferential during the resistance reaction, and precedes thedevelopment of the HR.

It is known that Pseudomonas solanacearum, a vascular bacterium, causesa lethal wilting of different plant species including Solanaceae. Inthis bacterium, a hypersensitive response (hrp) and pathogenicity genecluster has been shown to control both the ability to elicit the HR onnon-host plants and to cause the disease on host plants. In particular,hrp gene mutants of P. solanacearum have lost the ability to elicit anHR on tobacco plants. Recently, it was established that the hrpN gene ofthe hrp gene cluster of another bacterial pathogen, Erwinia amylovora,encodes a proteinaceous HR elicitor called harpin. This result confirmsthe important role of hrp genes in eliciting the HR. Upon infiltrationof tobacco leaves by an HR-inducing incompatible isolate, six differentgene families were characterized which are activated early during theinteraction, before any necrosis of the leaf was detected. These geneswhich were not induced upon infiltration by an hrp-isolate differed bythe accumulation levels of their transcripts during the incompatibleversus the compatible interactions: the str (sensitivity-related) genesare expressed to a similar extent in both types of interactions, whereasthe hsr genes are activated preferentially during the HR.

The present invention relates to an hsr gene family represented by agene, hereinafter designated hsr203J, the sequence of which is depictedin SEQ ID No. 1. The putative protein product (SEQ ID No. 2) of the geneexhibits little, if any substantial homology with known proteins. Testsemploying i.a. the promoter region of the hsr203J structural geneoperably linked to a reporter gene in transient gene expression assaysand in transgenic plants indicate that the expression of the hsr203Jgene is closely related to the development of hypersensitivity: thepromoter is specifically activated during the HR several hours beforethe appearance of the necrosis, and the localization of its activationis restricted to cells inoculated with an incompatible bacterialisolate.

According to the present invention there is provided a recombinant DNAsequence including a region comprising the nucleotide sequence depictedin SEQ ID No. 1 or a functional equivalent thereof, or a recombinantsequence comprising a part of said region or said equivalent.

Hereinafter where the term "functional equivalent" is used in respect ofthe protein encoding region of the DNA sequence the term means the saidregion wherein one or more codons have been replaced by their synonyms,ie codons which specify a corresponding amino acid or a correspondingtranscription termination signal.

Where the term "functional equivalent" is used in respect oftranscriptional regulatory regions of the sequence the term means thesaid region wherein one or more nucleotides have been replaced bydifferent nucleotides and/or the region wherein one or more nucleotideshave been added or removed with the proviso that the thus producedequivalents retain transcriptional regulatory activity and exhibitsubstantial homology with the region, or part thereof, which is 5'to theabove mentioned protein encoding region.

As used herein, the term "substantial homology" refers to a DNA sequencewhich hybridizes under conventional hybridization conditions with areference sequence. Preferably the hybridization conditions refer tohybridization in which the TM value is between 35 and 45° C. Mostpreferably the term substantial homology refers to a DNA sequence whichhybridizes with the reference sequence under stringent conditions (asdefined below).

The term "regulatory region" as used herein refers to the nucleotideregion in the sequence depicted in SEQ ID No. 1 which is 5'to theprotein encoding region in the sequence. The regulatory region thusincludes the promoter of the hsr203J gene and the functional componentsof the promoter which affect transcription. Such functional componentsinclude a "deletion promoter" and transcriptional "silencers" and"enhancers".

A "deletion promoter" within the context of the present invention is anyhsr203J derived promoter which has a deletion relative to the naturalpromoter and which still retains promoter activity. Such promoteractivity may be enhanced or substantially the same when compared to thenative promoter. The skilled man is aware of the manner in whichdeletion promoters can be assayed for retention of their promoteractivity. Deletion promoters according to the present invention areinducible, inter alia, by plant pathogens, and find utility inconstructs comprising structural genes providing for improved diseaseresistance.

Where the term "functional equivalent" is used in connection with aprotein, the sequence of which is dictated by at least a part of the DNAsequence depicted in SEQ ID No. 1, the term means a protein having alike function and like or improved specific activity, and a similaramino acid sequence. The present invention includes pure proteins whichhave an amino acid sequence which is at least 60% similar to thesequence or part (see below) thereof of the protein depicted in SEQ IDNo. 2. It is preferred that the degree of similarity is at least 60%,more preferred that the degree of similarity is at least 70% and stillmore preferred that the degree of similarity is at least 80%.

In the context of the present invention, two amino acid sequences withat least 60% similarity to each other are defined by having at least 70%identical or similar amino acids residues in the same position whenaligned optimally allowing for up to 4 deletions or up to 10 additions.

For the purpose of the present invention:

Alanine, Serine and Threonine are similar;

Glutamic acid and Aspartic acid are similar;

Asparagine and Glutamine are similar;

Arginine and Lysine are similar;

Isoleucine, Leucine, Methionine and Valine are similar;

Phenylalanine, Tyrosine and Tryptophan are similar.

Where the term "part" is used in connection with a protein sequence, theterm means a peptide comprised by the sequence depicted in SEQ ID No. 2and having at least 5 amino acids. More preferably the peptide has atleast 20 amino acids, and still more preferably the peptide has at least40 amino acids.

Where the term "part" is used in connection with a nucleotide sequence,the term means a nucleotide sequence comprised by the sequence depictedin SEQ ID No. 1 and having at least 15 nucleotides. More preferably thepart has at least 25 nucleotides, and still more preferably the part hasat least 40 nucleotides.

The invention also includes a recombinant DNA sequence including aregion comprising nucleotides 1413 to 2417 of the sequence depicted inSEQ ID No. 1 or a functional equivalent thereof, or a recombinantsequence comprising a part of said region or said equivalent.Nucleotides 1413 to 2417 correspond to the protein-encoding region ofthe hsr203J gene which is useful in that the gene product has afunctional role in regulating or providing for disease resistance inplants. Thus, the protein coding sequence, or a part thereof, of thehsr203J gene may be fused to an inducible promoter such as thatregulating expression of WIN, WUN or PR-proteins so that upon infectionby a compatible pathogen, expression of the hsr203J structural gene isinduced. The ensuing activation of the hypersensitive response by thehsr203J protein in infected plant cells halts further spread of thepathogen.

The invention also includes a recombinant DNA sequence including aregion comprising nucleotides 1 to 1341 of the sequence depicted in SEQID No. 1 or a functional equivalent thereof, or a recombinant sequencecomprising a part of said region or said equivalent. Nucleotides 1 to1341 correspond to the non-protein encoding region of the sequence whichis 5'to the said protein encoding region. The region of the said DNAsequence comprising nucleotides 1 to 1341 includes the transcriptionalregulatory region of the hsr203J gene, including the promoter (bindingsite for RNA polymerase) and transcriptional silencers and enhancers.

Silencer and enhancer elements are useful in that they enable modulationof the level of expression of the structural genes under their control.

The invention still further includes a recombinant DNA sequenceincluding a region comprising nucleotides 1 to 651 of the sequencedepicted in SEQ ID No. 1 or a functional equivalent thereof, or arecombinant sequence comprising a part of said region or saidequivalent. The region comprising nucleotides 1 to 651 includes atranscriptional silencer.

The invention still further includes a recombinant DNA sequenceincluding a region comprising nucleotides 652 to 1341 of the sequencedepicted in SEQ ID No. 1 or a functional equivalent thereof, or arecombinant sequence comprising a part of said region or saidequivalent. The region comprising nucleotides 652 to 1341 includes atranscriptional enhancer and the promoter (ie RNA polymerase bindingsite) of the hsr203J gene.

The invention further provides the use of hsr203J promoter sequences asaffinity substrates for the identification and subsequent purificationof hsr203j promoter binding proteins (hsr-PBP's) and proteins associatedwith these hsr-PBP's. Such hsr-PBP's have been partially characterized,are probably present constitutively and may bind to hsr203J promotersequences upon incompatible reaction of the host plant such as occurswhen Nicotiana tabacum L. is inoculated with specific strains ofPseudomonas solanacearum.

The invention still further includes a recombinant DNA sequenceincluding a region comprising nucleotides 1195 to 1341 of the sequencedepicted in SEQ ID No. 1 or a functional equivalent thereof, orrecombinant sequence comprising a part of said region or saidequivalent. The region comprising nucleotides 1195 to 1341 includes abacterial response element which is capable of binding to specificproteins which are produced by pathogens during their infection oftissue, and which are implicated in the development of thehypersensitive response (see above).

The invention still further includes a recombinant DNA sequenceincluding a region comprising nucleotides 1195 to 1268 of the sequencedepicted in SEQ ID No. 1 or a functional equivalent thereof, or arecombinant sequence comprising a part of said region or saidequivalent. This region more precisely defines the bacterial responseelement.

The invention still further includes a recombinant DNA sequence asdisclosed above wherein the said region, part or equivalent thereof islocated on the 5'side of, and is operably linked to, a protein-encodingsequence of a heterologous gene or to a sequence comprising nucleotides1413 to 2417 of the sequence depicted in SEQ ID No. 1 or a functionalequivalent thereof. It is particularly preferred that a translationenhancing sequence is present between the region or part or equivalentthereof, and the protein-encoding region of the DNA sequence 3' thereto.

The heterologous gene may be any suitable structural gene, including aselectable or screenable marker gene or a gene, the product of which iscapable of conferring resistance or tolerance to at least one of thefollowing: insects, herbicides, fungi, bacteria and viruses, a markergene for use in disease pressure forecasting and anti-feedant genes.

The promoter, and/or regulatory regions of the hsr203J gene may be fusedto a structural gene encoding a non-diffusible cytotoxic gene productsuch as an ribonuclease, protease, lipase or glucanase. Induction ofexpression of such structural genes provides a rapid and localizedresponse to infection by pathogens, and may be useful in providingresistance or improving tolerance of the plant to the pathogen.

Moreover, the regulatory regions of hsr203J gene may be used in thecreation of "detector" plants enabling the early detection of diseasepressure. The hsr203J promoter and/or regulatory regions thereof, may befused to a nucleotide sequence providing for a visual alteration to thehost plant phenotype upon activation of the promoter by infection. Suchsequences include the anti-sense orientation of the gene encoding theSmall Subunit of Ribulose B-phospho Carboxylase (SS-RUBISCO) whichcauses localized bleaching of green tissues. Such sequences could alsoencode a gene encoding a key enzyme in pigment biosynthesis such aschalcon synthase.

The invention also includes recombinant DNA according to the invention,which is modified in that codons which are preferred by the organisminto which the recombinant DNA is to be inserted are used so thatexpression of the thus modified DNA in the said organism yieldssubstantially similar protein to that obtained by expression of theunmodified recombinant DNA in the organism in which the protein-encodingcomponents of the recombinant DNA are endogenous.

The invention still further includes a DNA sequence which iscomplementary to one which, under stringent conditions, hybridizes toany one of the above disclosed recombinant DNA sequences.

"Stringent hybridization conditions" are those in which hybridization iseffected at between 50° and 60° C. in 2×saline citrate buffer containing0.1% SDS followed by merely rinsing at the same temperature but in abuffer having a reduced SCC concentration which will not affect thehybridizations that have taken place. Such reduced concentration buffersare respectively (a) 1×SCC, 0.1%SDS; or (b) 0.5×SCC, 0.1%SDS; or (c)0.1×SCC, 0.1%SDS.

The invention still further includes a DNA vector comprising arecombinant DNA sequence according to the invention or a DNA sequencewhich is complementary to one which, under stringent conditions,hybridizes thereto.

It is preferred that the vector according to the invention be used totransform a eukaryotic host, preferably of plant origin. It will beappreciated that suitable micro-organisms may be transformed with such avector, and such micro-organisms represent yet a further embodiment ofthe invention.

The term "plant" is used herein in a wide sense and refers todifferentiated plants as well as undifferentiated plant material such asprotoplasts, plants cells, seeds, platelets etc. that under appropriateconditions can develop into mature plants, the progeny thereof and partsthereof such as cuttings and fruits of such plants.

Preferred vectors will of course vary depending on the chosen host. Fordicotyledons, the vector may be introduced into a protoplast bycontacting the vector with the protoplast in a suitable medium and underappropriate conditions which render the protoplast competent for theuptake of DNA; the vector may also be employed in the form of anAgrobacteriurn tumefaciens Ti-plasmid derivative which infects plantcells or protoplasts. Monocotyledons are preferably transformed bymicro-injection, electroporation or by use of the micro-projectile gun,using the so-called ballistic technique. In any case, appropriatetransformation vectors and protocols are well known in the art. Thetransformed cells or protoplasts are cultured in an appropriate culturemedium, and a transformed plant is regenerated in a manner known per se.The introduced nuclear material is stably incorporated into the genomeof the regenerated transformed plants which accordingly express thedesired genes.

Examples of genetically modified plants according to the presentinvention include: fruits, including tomatoes, peppers, mangoes,peaches, apples, pears, strawberries, bananas, and melons; field cropssuch as canola, sunflower, tobacco, sugar beet, small grain cereals suchas wheat, barley and rice, corn and cotton, and vegetables such aspotato, carrot, lettuce, Brassica oleracea such as cabbage and onion.The particularly preferred plants are sugar beet and corn.

The invention still further includes the progeny or seeds of suchplants, and the seeds and progeny of said progeny.

The invention still further includes protein obtained by expression ofthe recombinant DNA according to the invention, and in particular,expressed protein having the amino acid sequence depicted in SEQ ID No.2, or a part thereof or a functional equivalent of said sequence orpart.

The Invention will be further apparent from the following description,and the associated Figures and Sequence Listings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) shows a chimeric construct used for transient gene expressionassays in tobacco protoplasts and for transformation of tobacco plantsvia Agrobacterium tumefaciens. Specifically the restriction map of thechimeric β-glucuronidase gene on pHG21 (or pHG21A) is depicted. The geneconsists of a translational fusion between 1.4 kb of the 5'flankingsequence from the hsr203J gene and the coding region of uidA gene linkedto the nopaline synthase gene polyadenylation signal (nos T).

FIG. 1(B) shows the sequence (SEQ ID No. 2) of the pHG21 translationalfusion joint. The hsr 203J gene sequence is in bold type and the uidAsequence is in standard type. The orientation is 5'to 3', and the arrowindicates the position of the fusion between the sequences.

FIG. 2 shows the effect of infection with different isolates (hrp, K 60and GMI 1000) of Pseudomonas solanacearum on hsr203J promoter activityin transformed tobacco protoplasts. As a control, water was added to theprotoplasts. Plasmids pBI201 and pBI221 are respectively negative andpositive control plasmids; pHG21 is the hsr203J-uidA gene fusion. GUSactivity assays were performed 24 h after incubation. The data shownrepresent the mean of three separate experiments.

FIG. 3 shows the time course of hsr203J promoter activation of the GUSgene in transgenic tobacco leaves infiltrated with different isolates(hrp, K 60 GMI 1000) of P. solanacearum. GUS activity was measured inextracts of four leaves from two pHG21-14A transformants.

FIG. 4(A) shows the induction of β-glucuronidase (GUS) activity in thebacterial-inoculated third leaf, and in the upper and lowerun-inoculated of transgenic tobacco leaves.

FIG. 4(B) shows the induction of β-glucuronidase activity in and aroundthe lesion of the inoculated third leaf. The following tissue sampleswere assayed: lesion meaning necrotic tissue resulting from the woundingand/or bacterial infection; 0-3 mm meaning apparently healthy tissue upto 3 mm from the lesion; 3-6 mm meaning apparently healthy tissue 3 to 6mm surrounding the lesion. Inoculation was performed on pHG21-14Atransformants. Small leaf perforations were covered by a droplet of thebacterial suspension (3 μL containing 10⁸ c.f.u./mL) or water, asindicated on the Figure. Tissue samples were collected 18 h afterinoculation.

FIG. 5(A) shows the effect of hrp mutants on the activation of hsr203Jpromoter in transgenic pHG21(14A) tobacco plants. Specifically depictedis the localization of hrp mutations in the different transcriptionunits of the hrp gene cluster. FIG. 5(B) shows measurements of GUSactivity in leaves at 18 h after inoculation by the hrp K60 or GMI 1000isolates or by water, or by the hrp mutants indicated in FIG. 5(A).Inoculation was performed as described for FIGS. 4(A) and 4(B).

FIG. 6 shows schematically the construction of plasmids pHGD havingseveral deletions of pHG21.

FIG. 7 shows in transgenic tobacco plants the expression of the GUS geneby constructs obtained by 5' promoter deletions of pHG21 (according tothe scheme of FIG. 5(A) and 5(B)). The plants were transformed with 5 μgDNA, and the value 100 was given to the GUS activity obtained bytransformation with the pHG21 construct. The Figure shows the increasein activity (after 18 hours) of the GUS gene as a consequence ofinfiltration of the transformed plants with the bacterial strains Delta3, K60 and GMI 1000. As controls plants were infiltrated with water.

OF THE SEQUENCES

SEQ ID No. I shows the nucleotide sequence of the hsr203J gene,including the protein encoding region and promoter and transcriptionalregulatory elements therefor, isolated from tobacco. The protein codingregion of the gene is comprised by nucleotides 1413 to 2417 in thesequence. Putative polyadenylation signals are present 3' to the proteincoding region of the gene and the sequence responsible for the HR iswithin about 1.4 kb of the 5' non-coding region of the gene. In essencethe sequence comprises:

a) a 72 bp mRNA leader sequence, located at nucleotides 1341 to 1412inclusive;

b) CAAT and TATA consensus sequences located at nucleotide positions1282-1286 and 1313-1316 respectively;

c) the translation start site codon at nucleotide positions 1413-1415;

d) the "deletion promoter" sequence located at nucleotides 1-1341inclusive which is substantially responsible for the promoter activity;

e) the sequence located at nucleotide positions 1195-1268 having anenhancing effect on the promoter activity;

f) the sequence located at nucleotide positions 1-651 having a silencingeffect on the promoter activity.

SEQ ID No. 2 shows the translation product of the hsr203J structuralgene, encoded by nucleotides 1413-2417 in SEQ ID No. 1;

SEQ ID No. 3 shows a linker region for a chimeric gene comprising the 5'flanking region of the hsr203J structural gene and the coding region ofthe uidA reporter gene. The start codon for the hsr203J structural geneis at nucleotides 10-12 in the sequence and nucleotides 13-64 encode theN-terminal sequence of the hsr203J gene product.

Bacterial Strains and Plant Material

The source of the Pseudomonas solanacearum strains used herein isdepicted in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Pseudomonas solanacearum wild type and mutant strains used in this            study,                                                                        and their ability to induce symptoms on tobacco                               Strains  Source or reference                                                                         Isolated from                                                                        Tobacco response                                __________________________________________________________________________    Wild type                                                                     GMI 1000 Boucher et al. (1)                                                                          Tomato HR                                              K60      Lozano et al. (17)                                                                          Tomato Disease                                         Mutants derived from GMI 1000 (deletion of hrp gene cluster)                  Δhrp                                                                             Boucher et al., unpublished                                                                        No symptoms                                     Mutants in hrp gene cluster derived from GMI 1000 (Tn5-B20 mutagenesis)       GMI 1462, 1475,                                                                        Arlat et al. (18)    No symptoms                                     1494, 1492, 1487                                                              GMI 1423, 1425                                                                         Arlat et al. (18)    Partial and/or                                                                delayed HR                                      Mutant derived from GMI 1000 (Tn5-B20 mutagenesis outside the hrp gene        cluster)                                                                      GMI 1485 Arlat et al. (18)    HR                                              __________________________________________________________________________

The GMI1000 and K60 isolates are wild-type P. solanacearum strains, theformer induces the development of an HR on tobacco leaves within 24 hafter infiltration, and the latter causes the typical lethal wiltingdisease. A derivative of the GMI 1000 isolate, called Δhrp, deleted forthe hrp gene cluster, causes no apparent symptoms in inoculated leaves.Eight mutant strains derived from GMI1000 by transposon Tn5-B20mutagenesis were used as described below. The GMI1462, 1475, 1494, 1485,1423 and 1425 strains are each mutated in one of the six putativetranscription units of the hrp gene cluster. All these strains have lostthe ability to cause an HR on tobacco, except strains GMI1423 and 1425which are mutated in the right-hand end of the hrp gene cluster, andinduce only a partial and/or a delayed HR on tobacco; and the strainGMI1485 which is mutated outside of the hrp gene cluster and elicits anormal HR on tobacco and constitutively expresses the structural gene ofβ-galactosidase. All these are grown at 28° C. in B or BGT media (1).The cultivars of Nicotiana tabacum L. used herein: Bottom Special andSamsun, exhibit similar responses after bacterial inoculation. Theseedlings are grown in vitro on Murashige and Skoog (MS) medium (2)during 4 to 5 weeks (25° C., 16 h photoperiod, 15 Watt/m²), and thentransferred to soil in a growth chamber (25° C., 16 h photoperiod, 30Watt/m²).

Isolation of hsr203J gene, and nucleotide sequence analysis

A tobacco (Nicotiana tabacum L. cultivar NK326) genomic libraryconstructed in the bacteriophage λ-Emb13 (Clontech) is screened with thepNt203 cDNA clone (3). The PstI insert of pNt203 is labeled by therandom primer technique (4). Replicate nitro-cellulose filters of thegenomic library are treated and hybridized as suggested by themanufacturers (Amersham). Four different genomic clones includinghsr203J are isolated. Exonuclease III deletions are performed at bothends of DNA inserts sublconed in the phagemid pKS (Stratagene) accordingto Henikoff (5), and both strands are sequenced by the dideoxy chaintermination method (6) using Sequenase (US Biochemical, Corp.). Sequencecompilation and analysis are performed by using the Genetics ComputerGroup software of the University of Wisconsin (7). Homology searcheswith the Genebank (release 71.0) and Swissprot (release 21.0) data basesare performed using the FASTA algorithm (8). The protein sequences areanalysed for potential N-terminal signal sequences and membrane-spanningdomains using release 5.0 of the PC/Gene Programme (Department ofMedical Biochemistry, University of Geneva, Switzerland). Thetranscription start site is determined by the primer extension techniqueusing polyA+RNA extracted from tobacco leaves 9 hours after inoculationwith the incompatible isolate and an oligonucleotide located at the ATGcodon (nucleotides 1413 to 1415 in SEQ ID No. 1).

Reporter gene constructs

A 2.2 kilobase (kb) BglII fragment containing 1.3 kb of the 5'non-coding region of the tobacco hsr203J gene and 890 base pairs (bp) ofthe nucleotide sequence downstream of the transcription start site iscloned into the BamHI site of phagemid pKS, to produce pKJ2.2. Thisplasmid is digested with BstBI, which cuts once 55 bp 3' of the hsr203Jtranslation initiation codon, and the BstBI generated ends were bluntend ligated by the Klenow fragment of DNA polymerase before digestionwith SalI. This 1.5 kb SalI--BstBI fragment is cloned into theSalI--SmaI site of the β-glucuronidase (GUS) expression binary vectorpBI101.2 (9) to produce to hsr203J--uidA gene fusion pHG21A. A 3.5 kbHindIII--EcoRI DNA fragment of pHG21A, including the hsr203J promoterand the uidA coding sequence, is ligated into the HindIII--EcoRIdigested pUC19 vector to produce pHG21, for transient expression geneassays (FIG. 1). The pHG21 and pHG21A constructs therefore contain 1341bp 5'non coding sequence, the 72 bp leader sequence, the first 55 bp ofthe coding sequence of hsr203J fused in frame with the GUS codingsequence, and the nopaline synthase (nos) gene polyadenylation signal.The translational fusion is confirmed by direct double-strandedsequencing with a GUS specific primer (10). Two additional plasmids,pBI201 and pBI221, contain respectively a promoterless uidA gene, and acauliflower mosaic virus (CaMV) 35S promoter--uidA gene, upstream of thenos terminator, in the pUC19 vector (Clontech).

Protoplast isolation and transient expression assays

Leaves of 4 to 5-week-old in vitro grown tobacco plants, cultivar SamsunNN, are used for isolation of protoplasts by incubating leaf sections inTO medium (11) containing 1 g/L cellulase R10 Onozuka, 200 mg/Lmacerozyme Onozuka (Yakult Honsha, Nishinomiya, Japan) and 500 mg/Lpectolyase Y23 (Seishin Pharmaceutical Ind.), for 15 h at 22° C. indarkness. Protoplasts are separated from the cellular debris by sievingthrough an 85 μm nylon mesh followed by centrifugation at 50 g for 5 minonto a 1 mL cushion of 19 % (w/v) sucrose. Floated protoplasts arewashed once with TO medium, counted, and adjusted to the density of1.5×10⁶ protoplasts/mL. Transformation is performed by incubating theprotoplasts (320 μL samples) at 45° C. for 5 min, after a brief coolingat room temperature, by adding plasmid DNA (50 μg per assay in 10 mMTris-HCl, pH 8) and 160 μL of a PEG solution (40% PEG, 0.4 M mannitol,30 mM MgCl₂, 0.1% Mes pH 5.8). Protoplasts are gently mixed for 10 minat room temperature. They are then collected by centrifugation andresuspended in 500 μL TO medium. The bacterial suspension (10bacteria/protoplast)prepared as previously described (12) is then added.After incubation at 28° C. for 24 h, the protoplasts are lysed by theaddition of 50 μL of 10×GUS buffer, centrifuged and the supernatant isassayed for GUS activity (10).

Transgenic tobacco plants

pHG2A, pBI121, and pBI101 are mobilised from Escherichia coli DH5α intoAgrobacterium tumefaciens strain LBA 4404 (13) and transgenic tobaccoplants (N. tabacum, Bottom Special) are generated by the leaf diskmethod (14). Transformed plants are selected on MS medium containing 0.8% Difco agar, kanamycin at 100 μg/mL and carbenicillin at 500 μg/mL.Transgenic plants are self-fertilized and seeds are collected. Theirgenotypes are determined by progeny (T2) analysis, by germination on MSmedium containing kanamycin (500 μg/mL).

Inoculation of transgenic plants with bacterial isolates

All the inoculation experiments are performed on kanamycin-resistant T2plants, with at least 2 plants of the same genotype per experimentalcondition. For the screening of transformants and kinetic experiments,tobacco leaves are detached from 8 week-old plants and infiltrated invacuo with the bacterial suspension (10⁷ c.f.u./mL) or water asdescribed in ref (12). Syringe infiltration experiments are performed on8 week-old plants by infiltrating the bacterial suspension (10⁸c.f.u./mL) into a small region of undetached leaves with a syringewithout a needle. For some experiments, inoculations were performed on 5week-old plants grown in Magenta cubes (Sigma) on MS medium. Each halfof a leaf is perforated 6 times with an 10 μL-Hamilton needle and a 3 μLdroplet of bacterial suspension (10⁸ c.f.u./mL in 0.4% Difco agar) isimmediately deposited at the wounded sites.

For localized root inoculation, 4 week-old plants are grown on a raft(Sigma) in contact with MS medium containing 0.2% Difco agar, andinoculated with a 3 μL, droplet of bacterial suspension through a woundmade with a needle at one centimeter from the root apex or at asecondary root emergence. For generalized root inoculation, the wholeplant is detached carefully from the raft, avoiding wounding, and theroot system is immersed in 7 μL of the bacterial suspension (10⁸c.f.u./mL).

Inoculated plants are maintained at 28° C., and analysed either directlyor stored at -80° C. after incubation time.

GUS assays

Plant tissue is ground in liquid nitrogen, homogenized in 1×GUS buffer,centrifuged for 5 min at 10,000 g and the supernatant assayed for GUSactivity, as previously described (15). Protein concentration isdetermined using the Bradford dye reagent. GUS activity is expressed aspicomoles of 4-methylumbelliferone per min per mg of protein.Alternatively, histochemical assays are performed on fresh tissue usingX-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide, Clontech) orMagenta-gluc (Biosynth AG) as the substrate (10). For some experiments,samples are fixed in 0.3% formaldehyde/50 mM NaPO₄ buffer pH7, thencleared by boiling in ethanol and stored in ethanol 70%.

β-galactosidase assays

Following the GUS histochemical assay, some samples are equilibrated inZ' buffer (16) (100 mM NaPO₄ buffer pH 7.4, 10 mM KCl, 1 mM MgSO₄),fixed in 1.25% glutaraldehyde for 1 h in order to inactivate endogenousplant β-galactosidases, rinsed and stained at 28° C. with 0.8 mg/mLMagenta-Gal (Biosynth Ag) or X-gal in Z' buffer containing 5 mM K₃FeCN)₆ and 5 mM K₄ Fe(CN)₆, then cleared by boiling in ethanol andobserved by dark- or bright-field microscopy.

Characterization of hsr203J gene

The hsr203J gene is isolated by screening a genomic tobacco library withpNt203 cDNA clone. It belongs to a small multi-gene family consisting ofa minimum of 4 genes (see ref. 3) and at least 2 genes of this familycorresponding to 2 different cDNA clones (pNt203 and pNt239) areexpressed during the HR.

Sequence analysis of the 2.7 kb DNA region of hsr203J (SEQ ID No. 1)reveals a single open reading frame (ORF) with no intron and a potentialcoding capacity of 355 amino acids. The nucleotide sequence of the said2.7 kb region is identical to the pNt239 cDNA clone except for 2substituted bp (not shown). These mismatches are probably due to theisolation of the genomic and cDNA clones from different tobaccocultivars: the genomic clone is isolated from cultivar NK326 whereas thepNt239 cDNA clone is obtained from the cultivar Bottom Special. Thepredicted hsr203J structural protein (SEQ ID No 2) has a Mr. of 37.5 kDaand a theoretical isoelectric point of 5.17.

The transcription start site is mapped by primer extension to a position72 bp upstream of the putative translation initiation codon. Thepromoter and 5' -untranslated region exhibited no obvious sequencehomology to cis-elements already described in defense genes.

Transient expression of the hsr203J-uid A gene fusion in tobaccoprotoplasts

pHG21 plasmid is composed of a translation fusion between 1.4 kb of the5' flanking sequence from the hsr203J gene and the coding region of theuidA reporter gene, linked to the 3' untranslated region of the nopalinesynthase gene (FIG. 2). The plasmids pBI201 and pBI221 are usedrespectively as negative and positive controls in transient assays.

Initial experiments show that protoplast viability as quantified byEvans blue exclusion is not significantly altered in the presence ofbacteria at 10 to 100 bacteria per protoplast (data not shown).Subsequently experiments are performed with 10 bacteria per protoplast.At this bacterial density, the expression of GUS fused to the hsr203Jpromoter in response to GMI1000 isolate is 6-fold higher than inresponse to the controls (water or αhrp inoculation) (FIG. 3). Incomparison, inoculation with the compatible isolate, K60, led to a2-fold increase in enzyme activity. These levels of GUS activity have tobe compared with those measured in protoplasts transformed with the CaMV35S-uidA gene fusion (pBI221) which exhibit a high and almostconstitutive level after the various inoculation treatments (FIG. 3).

The results of transient assays therefore indicate clearly that thehsr203J promoter contains all the necessary elements for itspreferential activation by the HR-inducing bacterial isolate, and thatthis expression system perfectly mimics the plant/pathogen interaction.

Expression of hsr203J-uid A gene fusion in transgenic tobacco

In order to determine the spatial and temporal patterns of expression ofthe hsr203J promoter in planta, the hsr203J-uidA gene fusion istransferred to tobacco by leaf disk transformation. T2 plants resistantto kanamycin are used in all the experiments. Of 23 kanamycin resistanttransformants, 20 expressed the gene fusion and these all exhibit thesame overall pattern of expression: GUS activity is found maximal afterinfiltration with GMI1000, with a 2- to 90-fold stimulation over controlinfiltrations (water or Δhrp), and a 2- to 25-fold induction over K60infiltration, 18 hours after inoculation (not shown). These levels arecomparable to those obtained in transient experiments after inoculationby GMI 1000 or K60.

Based on this analysis, a transformant (pHG21-14A) which displays a90-fold stimulation of GUS activity after incompatible inoculationcompared to control infiltrations, and contains one insertion of thegene fusion per haploid genome, is selected. The presence of a nativegene fusion is checked by Southern analysis of genomic DNA (not shown).

Assay of extractable GUS activity and GUS histochemical localization areboth used to monitor the activity of the hsr203J promoter in differentorgans during plant development and in response to bacterialinoculation. No GUS activity was detected in 4, 7 or 15 day-oldpHG21-14A tobacco seedlings, either in healthy leaves, or in flowers offully grown plants (data not shown). These data indicate that thehsr203J promoter is strongly activated in leaves inoculated with theHR-inducing isolate, GMI1000, 18 h after infiltration, as indicated bythe screening of all the transformants obtained. A kinetic study isperformed on transformant pHG21-14A (FIG. 3), which shows that in leavesinfiltrated with GMI1000, GUS activity increases to a level 12-fold overcontrol values 6 h after inoculation, reaches a maximum of 200-foldstimulation at 9 h, and decreases to an intermediate level (80-foldinduction) upon longer incubations. Much lower levels are measured afterK60 infiltration, and undetectable levels of GUS activity were found inleaves infiltrated with water or the Δhrp isolate at any incubationtime.

Plants transformed with the promoterless construct pHI101 shownegligible levels of GUS activity. Moreover, plants transformed withpBI121, which contain a CaMV 35S-uid A gene fusion, show similar levelsof enzyme activity, whatever the nature of the inoculum (not shown).Thus the hsr203J-uid A gene fusion exhibits a distinct and specificpattern of activation upon bacterial inoculation of transgenic tobaccoplants that closely matches the in vivo pattern of accumulation ofhsr203J transcripts in infiltrated tobacco leaves (3). These resultsalso indicate that hsr203J promoter is early and specifically activatedduring an incompatible plant/pathogen interaction, and that itsinduction is hrp gene-dependent since the bacterial isolate which isdeleted of hrp genes is unable to activate the hsr203J promoter.

Localization of hsr203J-uidA activation in response to bacterialinoculation

Different inoculation tests are performed on transformants pHG21-14A inorder to localize precisely hsr203J promoter activation in response tobacterial inoculation; first, in tobacco leaves in order to investigatepromoter induction during a typical HR, and secondly, in roots, whichare the organs naturally infected by the bacteria.

Leaf inoculations

In order to test whether the hsr203J-GUS gene expression is local orsystemic, leaves of 5 week-old transgenic plants are inoculated withbacterial suspension droplets. After incubation for 18 and 70 hours, GUSactivity is determined in half of the inoculated leaf as well as inupper and lower leaves. The results show a 15-fold induction of thisactivity in the inoculated leaf, whereas very low levels are detected inthe lower and upper leaves (FIG. 4A). The other half of the inoculatedleaf is used for histochemical GUS assay. A narrow blue-stained regionis visualized 18 h and 70 h after inoculation with the HR-inducingbacterial isolate, surrounding the wounded area, which is restricted toa few cell layers and is localized very close to yellowing, probablydead, cells. The intensity of the staining increases 70 h afterinoculation. Only a few dispersed cells exhibit a hint blue stainingafter K60 inoculation; water or Δhrp isolate inoculations induce nodetectable GUS expression. Staining of transgenic plants harboring achimeric uidA gene under the control of the CaMV 35S promoter results inthe staining of the entire leaf, with no preferential staining aroundthe lesions, thus demonstrating the specific nature of the induction ofthe hsr203J promoter in this area. A more detailed localization of thisactivation during infection is provided by GUS activity measurements insmall squares surrounding the lesion, 18 h after inoculation (FIG. 4B).High levels of enzyme activity (48-fold stimulation over control values)are found only within the necrotic lesion itself after inoculation byGMI1000. No detectable enzyme activity is found in tissue up to 3 mmaway from the lesion.

In order to determine how early the hsr203J promoter is activated in theinoculated area, histochemical GUS localizations are performed on leavesof 8 week-old transgenic plants locally infiltrated by a syringe withthe bacterial suspensions or K60. As early as 6 h after inoculation bythe GMI1000 isolate at which time there is no visible tissue necrosis,the leaf infiltrated area shows a blue staining whose intensityincreases 9 h after inoculation. At later incubation time points, ayellow necrosis progressively appears, limited on its border by a thinblue area still located within the infiltrated part of the leaf.

These different experiments show clearly that hsr203J-GUS expression isconfined to a restricted area corresponding precisely to cell layersinfected by the HR-inducing isolate, GMI1000.

Root inoculations

Roots of transgenic plants grown on rafts are wounded and inoculatedwith a droplet of bacterial suspension. After 48 h incubation,histochemical localization of GUS activity is performed. Staining onlyobserved in roots infected by GMI1000 extends from the initiallyinoculated site to a 2 mm distance in the root. Cytological studiesindicate that hsr203J promoter activation appears not to be cell-typedependent (not shown). A generalized root inoculation is also performedby simply immersing the whole root system in a bacterial suspension. Inthis case, GUS activity is found in restricted regions of the roots,i.e. at the point of origin of secondary roots. Expression of the genefusion at this specific location has to be correlated with the existenceof preferential sites of bacterial entry into the host which have beenobserved along the emergence sheath of secondary roots. At thesespecific sites, a double staining of GUS activity and bacteria by usinga bacterial isolate containing a β-galactosidase fusion, shows a goodcorrelation between the activation of the hsr203J promoter and thepresence of bacteria. A superficial and intercellular bacterialcolonization of the root tips has also been observed and results in astrong activation of the hsr203 promoter in this part of the root.

Thus, the hsr203J-GUS gene fusion exhibits a distinct and specificpattern of activation in transgenic tobacco plants in response tobacterial infection and one which closely matches the pattern ofbacterial ingress into the plant.

Dependence of hsr203J-uidA activation on hrp genes

Different P. solanacearum strains mutated in one of the sixtranscription units of the hrp gene cluster (FIG. 5A) are used toinoculate transgenic plants (pHG21-14A) by the droplet method. Thesemutant strains have lost the ability to induce an HR on tobacco,although two of them, GMI1425 and GMI1423, lead to a partial or delayedHR. 18 h after incubation, no effect on GUS activity can be detectedwith 6 out of 7 tested mutants; only GMI1423 leads to an increase inenzyme activity comparable to that of the wild type strain, GMI1000(FIG. 5B). These data indicate that hsr203J activation requires almost awhole functional hrp gene cluster.

Until now, no plant gene has been identified which is specificallyimplicated in the perception of an incompatible pathogen, the transferof that signal throughout the cell or finally the programmed cell death(HR) which provides an efficient mechanism for the limitation andeventual elimination of the pathogen.

The gene hsr203J (SEQ ID No. 1) is the first hypersensitivity-relatedgene to be isolated, whose promoter exhibits a rapid, high-levellocalized and specific activation in response to an HR-inducingbacterial isolate.

Construction of deletions of the 5' promoter region of pHG21

Unidirectional deletions of the promoter of the chimeric gene have beenrealized starting from the 5' end according to Henikoff (5). For thatpurpose, plasmid pHG21 (FIG. 1) is linearised employing the restrictionenzymes ShpI and SalI, and then digested by exonuclease III.Constructions having successive deletions, each distant by ca. 200 pb,are selected. The localization of the 5' end of the deletion isdetermined by sequencing the region and comparison with the nucleotidesequence of the hsr203J gene (see FIG. 6).

Effect of deletions on gene expression of the chimeric gene intransgenic tobacco plants

50 μg plasmid DNA corresponding to the different deletions (FIG. 6) areintroduced by transformation into tobacco plants. The GUS activity ismeasured 18 hours after inoculation.

FIG. 7 shows the expression of the GUS gene by constructs obtained by 5'promoter deletions of pHG21 (according to the scheme of FIG. 5). Theplants were transformed with 5 μg DNA, and the value 100 was given tothe GUS activity obtained by transformation with the pHG21 construct.The Figure shows the increase in activity (after 18 hours) of the GUSgene as a consequence of infiltration of the transformed plants with thebacterial strains Delta 3, K60 and GMI 1000. As controls plants wereinfiltrated with water. These experiments indicate the presence of 2main regions having a regulatory effect of the deletion promoter of thehsr203J gene.

One or more elements situated in the 1-651 nucleotide region of SEQ IDNo. 1 are responsible for a diminution of the expression of the chimericgene, and elements situated in the second region (nucleotides 652-1268)exhibit a positive effect on the activation of the promoter of thehsr203J gene.

The study of the spatial and temporal patterns of promoter activation inroots and leaves of transgenic plants inoculated with Pseudomonassolanacearum, indicate that

the promoter is specifically activated during the HR several hoursbefore the appearance of the necrotic lesion

the localization of its activation is restricted to the few cell layersin contact with the bacteria

the promoter does not respond to various stress conditions and is veryweakly activated during compatible interactions

the promoter activation is strongly dependent on hrp (hypersensitiveresponse and pathogenicity) genes of Pseudomonas solanacearum. Thesegenes control the ability of the bacterium to elicit the HR in resistantor non-host plant and to cause the disease on the host plant.

In favour of a major role of the bacterial hrp genes in the activationof hsr 203J gene promoter, is the fact that the hsr 203 promoter isexpressed in response to an HR specific elicitor, harpin, product of oneof the hrp genes of Erwinia amylovora. In response to this polypeptide,the promoter is activated at similar levels to those observed with thecorresponding avirulent strain, but more rapidly. Other potentialinducers such as biotic and abiotic elicitors, resistance inducers, donot affect its expression. The generality of the specific expression ofhsr 203J during incompatible interactions with bacterial pathogens hasbeen demonstrated by testing other pathogens such as Pseudomonassyringae pv pisi/pseudomonas syringae pv tabaci, and Erwinia amylovora.

In addition the functional analysis of the cis elements responsible forthe transcriptional activation of the hsr 203J gene in response to theincompatible bacterial strain, has been initiated by generating a seriesof 5' deletions and analysis of these constructs by transient assay andin transgenic plants. The results reveal the presence of a distalsilencer element, and of two positive regulatory elements, one beingquantitative (nucleotides 655-770 in SEQ ID No. 1), the other one beingspecific for the response to the bacterium, between nucleotides 1195 and1268 of the SEQ depicted in SEQ ID No. 1.

These results indicate that the hsr 203J gene promoter exhibits new andoriginal characteristics of activation with regard to plant defensegenes studied so far; its spatial and temporal program of activationtogether with its specific induction during the HR underline theimportance of this gene as a molecular tool to study the establishmentand regulation of the HR. In addition, a 74 bp sequence element has beendefined as responsible for the inducibility of the promoter by theavirulent pathogen.

Although the invention has been specifically described with reference toactivation of the hsr203J promoter in response to challenge of Tobaccoplants with an incompatible pathogen, it will be appreciated that thepromoter may likewise be activated by challenge of other plantstransgenic for the gene with other pathogens, including certain virusesand certain fungi, indicating that specific expression of the hsr203Jpromoter is a general phenomenon of incompatible interactions betweenhost and pathogen which lead to the hypersensitive response.

Moreover, the nucleotide sequence comprised by positions 1195 to 1268 ofthe sequence depicted in SEQ ID No. 1 containing the bacterial responseelement binds to nuclear protein extracts from various sources (healthyplants, plants inoculated with Pseudomonas solanacearum strains:compatible, incompatible and the hrp- mutant, after different incubationtimes). Such binding may be estimated by retardation gel analysis using,for example, the 74 bp region and several sub-fragments thus enablingidentification of discrete sequences within the BRE region which areuseful in providing genetic constructs comprising inducible diseaseresistance genes.

REFERENCES

1. Boucher, C. A., Barbeds, P. A., Trigalet, A. P., & Demery, D. A.(1985) J. Gen. Microbiol. 131, 2449-2457.

2. Murashige, T. & Skoog, F. (19629 Physiol. Plant 15, 473-497.

3. Marco, Y. J., Ragueh, F., Godiard, L., & Froissard, D. (1990) PlantMol. Biol. 15, 145-154.

4. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13.

5. Henikoff, S. (1984) Gene 28; 351-359.

6. Sanger, F., Nicklen, S. & Coulson, R. (1977) PNAS. (USA) 74,5463-5467.

7. Devereux, J., Haebefii, P. & Smithies, O. (1984) Nucleic Acids Res.13, 387-395.

8. Pearson, W. R., and Lipman, D. J. (1988) PNAS. (USA) 85, 2444-2448.

9. Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. (1987) EMBO J. 6,3901-3907.

10. Jefferson, R. A. (1987) Plant Mol. Biol. Reporter 5, 387-405.

11. Chupeau, Y., Bourgin, J. P., Missonier, C., Doffon, N. & Morel, G.(1974) Compte-rendu a l'Acad emie des Sciences Paris 278, 1565-1568.

12. Ragueh, F., Lescure, N., Roby, D. & Marco, Y. (1989) Physiol. Mol.Plant Pathol. 35, 23-33.

13. Bevan, M. W., (1984) Nucleic Acids Res. 12, 8711-8721.

14. Horsch, R. B., Fraley, R. T., Rogers, S. G., Sanders, P. R., Lloyd,A. & Hoffmann, N. L. (1984) Science 223, 496-498.

15. Roby, D., Broglie, K., Cressman, R., Biddie, Pl, Chet, I., &Bfoglie, R. (1990) Plant Cell 2, 999-1007.

16. Teeri, T. H., Lehvaslaiho, H., Franck, M., Uotila, J., Heino, P.,Palva, E. T., Van Montagu, M. and Herrera-Estralla, L. (1989) EMBO J. 8,343-350.

17. Lozano, J. C. & Sequeira, L. (1970) Phytopathol. 60, 833-838.

18. Arlat, M., Gough, C. L., Zischek, C., Barbefts, P. A., Trigalet, A.,& Boucher, C. A. (1992) Mol. Plant Microbe Interact. 5, 187-193

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2778 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Tobacco                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1413..2417                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GGATCTTAATGTTAGTTTATCTCTTGTTTTGAATATTTGATCTTAATTATAATTTATCCA60                CCATAAATTTTATTTTCAAAGATCAAACTATTGATATGACATTTCACTTTTTTATCTTTA120               TGTTTGTAGAATCATTAGTGGTATTGACTCTTACCAATCATTTTTTTTTCTTTCTCACAC180               ATTTATATTCTTAAATTTTCTTAGTTATTGTTTAATAATTGGGTATTTTTTAATATTACA240               CGAAAAATTGATTAAAAAAATATTATTTGAGTAGAAAAATAGTTCAAATATAATATAAAC300               ATATATTATCGTGGGAGTATTTTTTTCTCAATTTCAACTCTTTATGCAGTCCACTTAATA360               TTACTTTTATTTTTTCTTGGTATTAGACATTATGGAGTGGTAATGTATTGCCAATACGGC420               TGATTCTTATGAAATTGATTTTATTAAACCTTCCTACATTTTTAATAATAATTTAATAGA480               CAAAATTTTATTAATTTTAAATATTAAATATTAAAAATTAGTAGCATATAAGGTATTATA540               GTCCAAAAAATAGCTTATTACAGTTACGTACTCTTCCTATGAGTTCTTTCGTTTAATAAT600               GTAGGGCTATTTTGATATATTAATATTGTATTTATGCTTTTATAATAATATAGGCTCTCT660               TTTTTCTATATGAATTTGGACAATATAATACATTTTCAAATTAAATTAGTATCAAATAAT720               TGTATTTTTGCTTTTTTAATAATTTATACGCATGAATTTCATAATCCAGCATATTATGCT780               AGAACTTTTCGTGTTTCAACTAAAATAATGACTATTTTTCAATGACGTTACAAACACTGA840               CTAATTTTTGATTGCAGTCCGAAAACTATCTAGTCTATGCTATTTTCACTTTTCTAAACT900               CCCTGCCACTGTATGCTTTCATTGGATTAACCTTTAACCACACAAATATTTTAAAGAGTA960               ATGTTTGACAGCGTAATTTGAAACATCTACTATGCCTCTGTATATAATATCTAATGTTTG1020              TTCGTAGACCAATATTCTAATTCCTCTCTTGTAGACTAAACGGGGCTGTAACTAACTAAC1080              CACCATAGTTATCTAAATTAGTGACCCTAGCGACCATTGATAATTTGATACTGATCATTG1140              ACTTCCACCAAATCTACTTTCTAAATGTGGACTGACTCATTATGAATTTGTGAGGAAAAT1200              ACTTTCCTAATGCTAGTGCTCTTCCCATTATCTAAACTCCAAAATTTTGTAAAATTCTTT1260              GAACCTTCCTTTAAACTACCACAAATTTTCTTATCCTTTCCTATCTCACCATTATAAATA1320              GCCACGCACATGCAAACCAAAGGTACACACTAAACAAACTTCATTCTTCAAATTACTGAT1380              TACTCGAAAAAAACACTTCAAACTTTGCCAAAATGGTTCATGAAAAGCAAGTG1433                     MetValHisGluLysGlnVal                                                         15                                                                            ATAGAGGAAGTATCCGGCTGGCTTAGAGTTTTCGAAGACGGTTCAGTA1481                          IleGluGluValSerGlyTrpLeuArgValPheGluAspGlySerVal                              101520                                                                        GACCGGACTTGGACCGGTCCACCCGAAGTCAAATTCATGGCCGAGCCA1529                          AspArgThrTrpThrGlyProProGluValLysPheMetAlaGluPro                              253035                                                                        GTCCCACCCCATGACTACTTCATCGACGGCGTTGCCGTCAAAGATGTA1577                          ValProProHisAspTyrPheIleAspGlyValAlaValLysAspVal                              40455055                                                                      GTCGCCGACGAAAAATCCGGCAGCCGTCTCCGCATCTACTTACCTGAA1625                          ValAlaAspGluLysSerGlySerArgLeuArgIleTyrLeuProGlu                              606570                                                                        CGAAACGACAATTCCGCCAGCAAGCTTCCCGTCATTCTTCACTTCCAA1673                          ArgAsnAspAsnSerAlaSerLysLeuProValIleLeuHisPheGln                              758085                                                                        GGCGGCGGCTTTTGTGTCAGCCATGCTGATTGGTTCATGTACTACACT1721                          GlyGlyGlyPheCysValSerHisAlaAspTrpPheMetTyrTyrThr                              9095100                                                                       GTCTACACGCGCCTAGCGCGCGCGGCCAAAGCTATCATTGTCTCCGTC1769                          ValTyrThrArgLeuAlaArgAlaAlaLysAlaIleIleValSerVal                              105110115                                                                     TTCCTCCCCCTCGCGCCGGAGCACCGCCTCCCAGCTGCCTGCGATGCC1817                          PheLeuProLeuAlaProGluHisArgLeuProAlaAlaCysAspAla                              120125130135                                                                  GGTTTCGCCGCTCTCCTCTGGCTCCGGGACCTCTCCCGGCAGCAAGGA1865                          GlyPheAlaAlaLeuLeuTrpLeuArgAspLeuSerArgGlnGlnGly                              140145150                                                                     CACGAGCCCTGGCTCAACGATTACGCAGATTTCAACCGAGTATTCCTC1913                          HisGluProTrpLeuAsnAspTyrAlaAspPheAsnArgValPheLeu                              155160165                                                                     ATCGGAGACAGCTCCGGCGGGAACATAGTCCACCAAGTTGCCGTCAAA1961                          IleGlyAspSerSerGlyGlyAsnIleValHisGlnValAlaValLys                              170175180                                                                     GCCGGCGAGGAAAACTTATCTCCAATGCGACTGGCCGGCGCAATTCCG2009                          AlaGlyGluGluAsnLeuSerProMetArgLeuAlaGlyAlaIlePro                              185190195                                                                     ATCCATCCAGGTTTCGTGCGGTCCTATCGGAGCAAATCGGAGCTAGAA2057                          IleHisProGlyPheValArgSerTyrArgSerLysSerGluLeuGlu                              200205210215                                                                  CAAGAGCAAACCCCGTTTTTAACATTAGATATGGTGGATAAATTTCTA2105                          GlnGluGlnThrProPheLeuThrLeuAspMetValAspLysPheLeu                              220225230                                                                     GGGTTAGCTTTACCAGTAGGGAGCAACAAGGATCATCAAATAACATGT2153                          GlyLeuAlaLeuProValGlySerAsnLysAspHisGlnIleThrCys                              235240245                                                                     CCGATGGGAGAGGCGGCGCCGGCAGTGGAGGAGCTTAAATTACCGCCT2201                          ProMetGlyGluAlaAlaProAlaValGluGluLeuLysLeuProPro                              250255260                                                                     TATTTGTACTGTGTGGCGGAGAAAGATCTGATAAAGGACACTGAAATG2249                          TyrLeuTyrCysValAlaGluLysAspLeuIleLysAspThrGluMet                              265270275                                                                     GAGTTTTACGAAGCTATGAAAAAGGGGGAAAAGGATGTAGAGCTGTTT2297                          GluPheTyrGluAlaMetLysLysGlyGluLysAspValGluLeuPhe                              280285290295                                                                  ATTAACAATGGAGTGGGACATAGCTTTTATCTTAACAAAATTGCTGTT2345                          IleAsnAsnGlyValGlyHisSerPheTyrLeuAsnLysIleAlaVal                              300305310                                                                     AGAATGGACCCTGTAACTGGTTCTGAAACTGAAAAACTTTATGAAGCC2393                          ArgMetAspProValThrGlySerGluThrGluLysLeuTyrGluAla                              315320325                                                                     GTTGCAGAGTTCATCAACAAGCATTAAAAGGAGAAAATTTGTGGTT2439                            ValAlaGluPheIleAsnLysHis                                                      330335                                                                        TTGCAGAATATTTGTTTGTTGCATGCATGTTCAAGATTTTGATGTACCGTCTTGATTGTC2499              ACGTTCTAATGGTTTTGTAATTATAATTATGAGGAGTAAATTTCTATTGTTGCGTAGAAA2559              TGTTTTTTCTTTGGTAGTAAATGTTTATTTGTAATACTTTAAAAAGTGGACAAATTTCTT2619              TTGAGATTCATGAAATAATATCTTTAAATTTCGAATGTCAATAAGTCCAGAAATTGAAAT2679              GTATCTGTACCGTCAATGAAGTCTCCTTGAGGCTTTTTTTCACATGATATCGTCTATACC2739              ACCAAAAAGTTTGATAAGCTATACAATATGAGATTCTCG2778                                   (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetValHisGluLysGlnValIleGluGluValSerGlyTrpLeuArg                              151015                                                                        ValPheGluAspGlySerValAspArgThrTrpThrGlyProProGlu                              202530                                                                        ValLysPheMetAlaGluProValProProHisAspTyrPheIleAsp                              354045                                                                        GlyValAlaValLysAspValValAlaAspGluLysSerGlySerArg                              505560                                                                        LeuArgIleTyrLeuProGluArgAsnAspAsnSerAlaSerLysLeu                              65707580                                                                      ProValIleLeuHisPheGlnGlyGlyGlyPheCysValSerHisAla                              859095                                                                        AspTrpPheMetTyrTyrThrValTyrThrArgLeuAlaArgAlaAla                              100105110                                                                     LysAlaIleIleValSerValPheLeuProLeuAlaProGluHisArg                              115120125                                                                     LeuProAlaAlaCysAspAlaGlyPheAlaAlaLeuLeuTrpLeuArg                              130135140                                                                     AspLeuSerArgGlnGlnGlyHisGluProTrpLeuAsnAspTyrAla                              145150155160                                                                  AspPheAsnArgValPheLeuIleGlyAspSerSerGlyGlyAsnIle                              165170175                                                                     ValHisGlnValAlaValLysAlaGlyGluGluAsnLeuSerProMet                              180185190                                                                     ArgLeuAlaGlyAlaIleProIleHisProGlyPheValArgSerTyr                              195200205                                                                     ArgSerLysSerGluLeuGluGlnGluGlnThrProPheLeuThrLeu                              210215220                                                                     AspMetValAspLysPheLeuGlyLeuAlaLeuProValGlySerAsn                              225230235240                                                                  LysAspHisGlnIleThrCysProMetGlyGluAlaAlaProAlaVal                              245250255                                                                     GluGluLeuLysLeuProProTyrLeuTyrCysValAlaGluLysAsp                              260265270                                                                     LeuIleLysAspThrGluMetGluPheTyrGluAlaMetLysLysGly                              275280285                                                                     GluLysAspValGluLeuPheIleAsnAsnGlyValGlyHisSerPhe                              290295300                                                                     TyrLeuAsnLysIleAlaValArgMetAspProValThrGlySerGlu                              305310315320                                                                  ThrGluLysLeuTyrGluAlaValAlaGluPheIleAsnLysHis                                 325330335                                                                     (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      TTTGCCAAAATGGTTCATGAAAAGCAAGTGATAGAGGAAGTATCCGGCTGGCTTAGAGTT60                TTCGGGGTAGGTCAGTCCCTTATGTTACGTCCT93                                           __________________________________________________________________________

We claim:
 1. Recombinant DNA including a region comprising a nucleotidesequence selected from the group consisting ofa) nucleotides 1 to 1341of the sequence depicted in SEQ ID No.1; b) nucleotides 1 to 651 of thesequence depicted in SEQ ID No.1; c) nucleotides 652 to 1341 of thesequence depicted in SEQ ID No.1; d) nucleotides 1195 to 1268 of thesequence depicted in SEQ ID No.1; and e) nucleotides 1195 to 1341 of thesequence depicted in SEQ ID No.1.
 2. DNA according to claim 1 whereinsaid region comprises nucleotides 1 to 1341 of the sequence depicted inSEQ ID No.1.
 3. DNA according to claim 1 wherein said region comprisesnucleotides 1 to 651 of the sequence depicted in SEQ ID No.1.
 4. DNAaccording to claim 1 wherein said region comprises nucleotides 652 to1341 of the sequence depicted in SEQ ID No.1.
 5. DNA according to claim1 wherein said region comprises nucleotides 1195 to 1341 of the sequencedepicted in SEQ ID No.1.
 6. DNA according to claim 1 wherein said regioncomprises nucleotides 1195 to 1268 of the sequence depicted in SEQ IDNo.1.
 7. Recombinant DNA of claim 1 wherein said nucleotide sequence isoperably joined on at least one end to a DNA fragment not naturallyassociated with said nucleotide sequence.
 8. Recombinant DNA including aregion comprising a nucleotide sequence selected from the groupconsisting ofI) nucleotides 1 to 1341; ii) nucleotides 1 to 651; iii)nucleotides 1195 to 1268; iv) nucleotides 1195 to 1341; and v)nucleotides 652 to 1341of the sequence depicted in SEQ ID No.1, whereinsaid region is located on the 5' side of, and operably linked to aprotein encoding sequence of a heterologous gene or to a proteinencoding sequence comprising nucleotides 1413 to 2417 of said sequencedepicted in SEQ ID No.1.
 9. DNA according to claim 8 wherein atranslation enhancing sequence is present between the region and theprotein encoding sequence.
 10. DNA according to claim 8 wherein theheterologous gene is a selectable or screenable marker or a gene, theproduct of which is capable of conferring resistance or tolerance toinsects, herbicides, fungi, bacteria or viruses.
 11. The sequenceaccording to claim 8, which is modified in that codons which arepreferred by the organism into which the recombinant DNA is to beinserted, are used so that expression of the modified DNA in saidorganism yields protein having the same amino acid sequence as thatobtained by expression of the unmodified recombinant DNA in the organismin which the protein-encoding components of the recombinant DNA areendogenous.
 12. A DNA vector comprising a sequence according to claim 8.13. A micro-organism which has been transformed with recombinant DNAaccording to claim
 8. 14. A protoplast which has been transformed withrecombinant DNA according to claim
 7. 15. A micro-organism which hasbeen transformed with a recombinant DNA according to claim
 11. 16. Aprotoplast which has been transformed with a recombinant DNA accordingto claim
 11. 17. A vector comprised of the recombinant DNA of claim 7.18. A plant cell comprised of the recombinant DNA of claim
 7. 19. Aplant comprised of the plant cells of claim 18.